Establishment Of Two-dimensional Electrophoresis System And Differential Proteomic Analysis Of Floral Bud Of Temperature Sensitive Male Sterile Line SP2S In Brassica Napus L

Thermo-sensitive male sterile in Brassica napus is one important way of heterosis utilization, the thermo-sensitive male sterile line SP2 S in Brassica napus was found and breeding from the offspring of a oilseed rape resource, which was showed fertile in low temperature in Yangling spring, and thoroughly abortion above the critical temperature. The anthers abortive began in pollen mother cell meiosis period. The methods of two dimensional electrophoresis and mass spectrometry were used to analyze the differential proteins between SP2 S and it’s near isogenic line SP2 F under the stage of meiosis and later stage of uni-nucleate. In order to get the genes involved in ferterility traits, this research studied its sterility mechanism from the aspects of proteomics.1.Establishment of two-dimensional electrophoresis system for foral bud proteome. Using TCA/acetone and Tris-acetone-phenol methods to extract total proteins from floral buds respectively, selecting and optimizing system of two dimensional electrophoresis in concentrations of polyacrylamide gel, sample loading amount, and pH of immobilized pH gradient gels. The results showed that clear background, well distributed protein spots and reproducible 2-DE maps could be acquired following TCA/acetone extraction, electrophoresis system including 10% SDS-PAGE, 450 μg of sample loading quantity, and 17 cm immobilized pH gradient(IPG) strip with pH 4~7, and modified staining using coomassie brilliant blue.2.Differential proteomic analysis of floral bud proteins of SP2 S. Using the optimized 2-DE system to get reproducible 2-DE maps between SP2 S and it’s near isogenic line SP2 F at the stage of pollen mother cell meiosis and later stage of uni-nucleate microspore. The results indicated that 99 differential spots were acquired using the analysis of software with PDQuest 7.4.0, which including 55 differential spots in the stage of meiosis and 44 differential spots in the monokaryotic stage. Among which 28 well reproducible spots were analyzed by MALDI-TOF-TOF mass spectrometry, and 23 were identified to be 12 kinds of proteins. They were amino acid metabolism, carbohydrate metabolism, photosynthesis, synthesis and degradation of protein, lipid metabolism, stress response, glycolysis, hydrolase, protease, oxidordeuctase, transferase and purple acid phosphatase. It concluded that the main impact factors were those proteins which about the formation of pollen grain and degradation of anther tapetum(glutamine synthetase, lipoxygenase and beta-glucosidase 20), and the poisoning caused by imbalance of protein metabolism and energy metabolism(elongation factor EF-2, coniferaldehyde/sinapaldehyde dehydrogenase and alcohol dehydrogenase).